Department of Biochemistry & Molecular Biology, Michigan State University, East Lansing, MI, USA.
Methods Mol Biol. 2024;2839:131-149. doi: 10.1007/978-1-0716-4043-2_8.
Heme o is an Fe-porphyrin involved in the majority of aerobic respiration pathways found in all three domains of life. In eukaryotes and most aerobic prokaryotes, heme o functions solely as the precursor for the synthesis of heme a, a necessary cofactor for most heme-copper terminal oxidases. In some prokaryotes, such as Escherichia coli (E. coli), heme o can serve as a cofactor for heme-copper oxidases instead of heme a. Given its role as a key substrate or cofactor, purified heme o promises to be a valuable resource for the study of heme-copper oxidase assembly and activity. However, commercially available heme o is sold in limited quantities at a relatively high cost (compared to the prototypical heme b), making the use of heme o purchased from suppliers unfeasible for such studies. In this chapter, we present step-by-step methods both for heme o isolation from E. coli overexpressing heme o synthase and for HPLC analysis of cellular hemes (i.e., heme o and heme b).
亚铁血红素 o 是一种含铁卟啉，参与了所有三个生命领域中大多数有氧呼吸途径。在真核生物和大多数需氧原核生物中，亚铁血红素 o 仅作为血红素 a 合成的前体，血红素 a 是大多数血红素-铜末端氧化酶的必需辅因子。在一些原核生物中，如大肠杆菌（E. coli），亚铁血红素 o 可以替代血红素 a 作为血红素-铜氧化酶的辅因子。鉴于其作为关键底物或辅因子的作用，纯化的亚铁血红素 o 有望成为研究血红素-铜氧化酶组装和活性的宝贵资源。然而，商业上可获得的亚铁血红素 o 的数量有限，价格相对较高（与典型的血红素 b 相比），这使得从供应商处购买的亚铁血红素 o 无法用于此类研究。在本章中，我们提供了从过表达亚铁血红素合酶的大肠杆菌中分离亚铁血红素 o 和 HPLC 分析细胞血红素（即亚铁血红素 o 和血红素 b）的分步方法。
